Methods for producing interfering RNA molecules in mammalian cells and therapeutic uses for such molecules

ABSTRACT

Methods for producing interfering RNA molecules in mammalian cells are provided. Therapeutic uses for the expressed molecules, including inhibiting expression of HIV, are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 14/164,625 filed 27 Jan. 2014, which in turn is a continuationof U.S. patent application Ser. No. 13/324,104 filed 13 Dec. 2011, whichin turn is a continuation of U.S. patent application Ser. No. 12/881,509filed 14 Sep. 2010, now U.S. Pat. No. 8,076,071, which in turn is adivision of U.S. patent application Ser. No. 10/365,643 filed 13 Feb.2003, now U.S. Pat. No. 7,820,632, which in turn relates to and claimspriority under 35 U.S.C. §119(e) to U.S. provisional patent applicationSer. No. 60/356,127, filed 14 Feb. 2002. Each application isincorporated herein by reference

REFERENCE TO GOVERNMENT SUPPORT

This invention was made with government support under Grant No. A1 29329awarded by the National Institutes of Health. The United Statesgovernment has certain rights in the invention.

SEQUENCE SUBMISSION

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is entitled1954577SequenceListing.txt, created on 5 Jan. 2015 and is 3 kb in size.The information in the electronic format of the Sequence Listing is partof the present application and is incorporated herein by reference inits entirety.

FIELD OF THE INVENTION

The present invention relates to RNA interference. More particularly,the present invention relates to methods for producing interfering RNAmolecules in mammalian cells, and genetic and therapeutic uses for suchexpressed molecules.

BACKGROUND OF THE INVENTION

RNA interference is the process of sequence-specific,post-transcriptional gene silencing in animals and plants initiated bydouble stranded (ds) RNA that is homologous to the silenced gene(Hammond, S. M. et al., 2000; Fire, A., 1999; Sharp, P. A., 2001). Inparticular, synthetic and endogenous siRNAs are known to direct targetedmRNA degradation (Hammond, S. M. et al., 2000; Elbashir, S. M. et al.,2001; Caplen, N. J. et al., 2001; Clemens, J. C. et al., 2000; Lipardi,C. et al., 2001; Elbashir, S. M. et al., 2001; Ui-Tei, K. et al., 2000).

This powerful genetic technology has usually involved injection ortransfection of ds RNA in model organisms. RNA interference also is apotent inhibitor of targeted gene expression in a variety of organisms(Wianny, F. et al., 2000; Kennerdell, J. R. et al., 1998; Fire, A. etal., 1998; Oelgeschlager, M. et al., 2000; Svoboda, P. et al., 2000).Recent studies by several groups (Lipardi, C. et al., 2001; Sijen, T. etal., 2001) suggest that ds small interfering RNAs (siRNAs) are part of ariboprotein complex that includes an RNAse III-related nuclease (Dicer)(Bernstein, E. et al., 2001), a helicase family (Dalmay, T. et al.,2001; Cogoni, C. et al., 1999), and possibly a kinase (Nykanen, A. etal., 2001) and an RdRP (Lipardi, C. et al., 2001; Smardon, A. et al.,2000). The mechanism proposed by Lipardi et al. (Lipardi, C. et al.,2001) is that one of the siRNA oligomers (antisense to the target RNA)primes an RdRP, generating longer dsRNAs, which are then cleaved by theRNAse III activity into additional siRNA duplexes, thereby amplifyingthe siRNAs from the target template.

dsRNA≧30 bp can trigger in mammalian cells interferon responses that areintrinsically sequence-nonspecific (Elbashir, S. M. et al., 2001).However, duplexes of 21-nucleotide (nt) siRNAs with short 3′ overhangscan mediate RNA interference in a sequence-specific manner in culturedmammalian cells (Elbashir, S. M. et al., 2001). Two groups havedemonstrated that 19 to 21 base duplexes with 3′UU or TT overhangs caneffectively elicit an siRNA response in mammalian cells (Elbashir, S. M.et al., 2001; Caplen, N. J. et al., 2001). However, one limitation tothe use of siRNA as a therapeutic reagent in vertebrate cells is thatshort, highly defined RNAs need to be delivered to target cells, whichthus far has been accomplished only by using synthetic, duplexed RNAsdelivered exogenously to cells (Elbashir, S. M. et al., 2001; Caplen, N.J. et al., 2001).

The present invention overcomes at least the above limitation.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides methods for producingdouble stranded, interfering RNA molecules in mammalian cells, andpreferably human cells, by introducing into the cells DNA sequencesencoding the interfering RNA molecules.

In another aspect, the method comprises a) inserting DNA sequencesencoding a sense strand and an antisense strand of an interfering RNAmolecule into a vector comprising a suitable promoter, preferably a RNApol III promoter, and b) introducing the vector into a mammalian cell sothat the RNA molecule can be expressed.

In a preferred embodiment, the present invention includes firstselecting a target sequence, which preferably is accessible to thepairing between the target sequence and interfering RNA required for theinterfering RNA to function properly. Methods for identifying targetsites may be carried out using synthetic DNA oligonucleotides in cellextracts and/or a site selection approach on native RNAs, as describedherein. Once an optimal target site has been identified, the appropriatesequences for making the sense and antisense strands of the interferingRNA molecule can be synthesized.

Possible target sites include those found on the transcription productsof cellular or infectious agent genes (viral, bacterial etc.).

In another preferred embodiment, the RNA molecule produced is a smallinterfering RNA (siRNA) molecule, while the DNA sequences encoding thesense and antisense strands of the siRNA are siDNA.

In another preferred embodiment, the RNA pol III promoter is a mammalianU6 promoter, and more preferably the human U6 RNA Pol III promoter.

In another aspect, the invention provides methods for inhibiting theexpression of target genes, comprising introducing one or more vectorsinto a mammalian cell, wherein the one or more vectors comprise asuitable promoter and DNA sequences encoding a sense strand and anantisense strand of an interfering RNA, preferably siRNA, molecule. Theinterfering RNA molecule, which preferably is specific for thetranscription product of the target gene, can be then expressed andinitiate RNA interference of protein expression of the target gene inthe mammalian cell, thereby inhibiting expression of the target gene.

In another aspect, the invention provides a method for testing theexpression and function of siRNA molecules, comprising co-introducinginto a mammalian cell i) one or more vectors comprising a suitable firstpromoter and DNA sequences encoding a sense strand and an antisensestrand of an siRNA molecule, and ii) a vector comprising a target geneand a suitable second promoter. The siRNA molecule can be then expressedand initiate RNA interference of expression of the target gene, therebypotentially inhibiting expression of the target gene. Thus, theendogenous expression and function of the siRNA molecule can be assayedbased on the presence, if any, of RNA interference and more particularlyby any inhibition of expression of the target gene.

The present invention thus provides many possible therapeuticapplications, based on the design of the siRNA molecules and theirspecificity for selected disease targets. For example, one applicationof the invention is the treatment of HIV, for which siRNA molecules maybe designed to inhibit the expression of selected HIV targets, thusinhibiting HIV expression.

In a preferred embodiment, the invention provides a method forinhibiting expression of an HIV target gene, comprising introducing oneor more vectors into a mammalian cell, preferably an HIV-infected humancell. The one or more vectors comprise a suitable promoter and DNAsequences encoding a sense strand and an antisense strand of an siRNAmolecule, which preferably is specific for the transcription product ofthe HIV target gene. More preferably, the siRNA molecule is specific fora selected target site on the transcription product of the selected HIVtarget gene. The siRNA molecule can be then expressed and initiate RNAinterference of expression of the target gene, thereby inhibitingexpression of the target gene.

In a more preferred embodiment, the HIV is HIV-1. In another preferredembodiment, multiple siRNA constructs targeted to different sites in theHIV genome may be expressed, thereby initiating RNA interference ofexpression of several different HIV target genes and thus possiblycircumventing genetic resistance of the virus.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A shows a schematic diagram of a target rev-EGFP construct.

FIG. 1B (SEQ ID NO:9) and 1C (AS(I) (SEQ ID NO:1), S(I) (SEQ ID NO:2),AS(II) (SEQ ID NO:3), S(II) (SEQ ID NO:4)) show schematic diagrams of aU6 promoter construct and a U6 promoter driven siRNA construct.

FIG. 1D shows siRNAs (S(I) (SEQ ID NO:5), AS(I) (SEQ ID NO:6), S(II)(SEQ ID NO:7), AS(II) (SEQ ID NO:8) in accordance with an embodiment ofthe invention.

FIG. 2 shows gel photographs from accessibility assays for sites I andII in cell extracts prepared from rev-EGFP expressing cells.

FIGS. 3A to 3J show photographs obtained from fluorescent microscopeimaging of the effect of siRNA on EGFP expression.

FIG. 4 is bar graph showing the extent of inhibition of EGFP expressionby siRNAs in accordance with an embodiment of the invention.

FIGS. 5A to 5G show autoradiographs of Northern gel analyses.

FIG. 6 is a graph showing inhibition of HIV-1 NL4-3 by siRNAs inaccordance with an embodiment of the invention.

DETAILED DESCRIPTION OF THE INVENTION

Interfering RNA molecules, and more preferably siRNA molecules, producedand/or used in accordance with the invention include those types knownin the art. The interfering RNA, and preferably siRNA, molecules aredouble-stranded (ds) RNAs that preferably contain about 19 to 23 basepairs. The molecules also may contain 3′ overhangs, preferably 3′UU or3′TT overhangs.

The term “introducing” encompasses a variety of methods of introducingDNA into a cell, either in vitro or in vivo, such methods includingtransformation, transduction, transfection, and infection. Vectors areuseful and preferred agents for introducing DNA encoding the interferingRNA molecules into cells. Possible vectors include plasmid vectors andviral vectors. Viral vectors include retroviral vectors, lentiviralvectors, or other vectors such as adenoviral vectors or adeno-associatedvectors.

In one embodiment, the DNA sequences are included in separate vectors,while in another embodiment, the DNA sequences are included in the samevector. If the DNA sequences are included in the same vector, the DNAsequences may also be inserted into the same transcriptional cassette.

Alternative delivery systems for introducing DNA into cells may also beused in the present invention, including, for example, liposomes, aswell as other delivery systems known in the art.

Suitable promoters include those promoters that promote expression ofthe interfering RNA molecules once operatively associated or linked withsequences encoding the RNA molecules. Such promoters include cellularpromoters and viral promoters, as known in the art. In one embodiment,the promoter is a RNA pol III promoter, which preferably is locatedimmediately upstream of the DNA sequences encoding the interfering RNAmolecule. Various viral promoters may be used, including, but notlimited to, the viral LTR, as well as adenovirus, SV40, and CMVpromoters, as known in the art.

In a preferred embodiment, the invention uses a mammalian U6 RNA Pol IIIpromoter, and more preferably the human U6snRNA Pol III promoter, whichhas been used previously for expression of short, defined ribozymetranscripts in human cells (Bertrand, E. et al., 1997; Good, P. D. etal., 1997). The U6 Pol III promoter and its simple termination sequence(four to six uridines) were found to express siRNAs in cells.Appropriately selected interfering RNA or siRNA encoding sequences canbe inserted into a transcriptional cassette, providing an optimal systemfor testing endogenous expression and function of the RNA molecules.

In a preferred embodiment, the mammalian cells are human cells. However,it is also understood that the invention may be carried out in othertarget cells, such as other types of vertebrate cells or eukaryoticcells.

In accordance with the invention, effective expression of siRNA duplexestargeted against the HIV-1 rev sequence was demonstrated. Using arev-EGFP (enhanced green fluorescent protein) fusion construct intransient co-transfection assays, ca 90% inhibition of expression wasobserved. The same siRNA expression constructs have been tested againstHIV in co-transfection assays resulting in a four-log reduction in HIVp24 antigen levels.

The above results were achieved using a human U6 snRNA Pol III promoterto express the appropriate 21 base oligomer RNAs in human cells. Thepromoter design is such that the first base of the transcript is thefirst base of the siRNA, and the transcript terminates within a run of 6U's encoded in the gene. The U6+1 promoter initiates transcription witha tri-phosphate, and the transcript is not capped unless the first 27bases of the U6 RNA are included in the transcript (Bertrand, E. et al.,1997; Good, P. D. et al., 1997). Thus, it was believed that siRNAs couldbe made whose structure would closely mimic certain predefinedrequirements (Elbashir, S. M. et al., 2001; Caplen, N. J. et al., 2001).

As stated above, expression cassettes are designed such that sequencesencoding sense and antisense strands of the siRNA can be in either thesame or separate vectors. Although the vector containing both sense andantisense strands was predicted to be superior to co-transfecting thetwo separately, this was not the case. It is likely that theco-transfection juxtaposes the two sequences so that transcripts haveample opportunity to form dsRNAs. An interesting feature of theexpression system is that in cells expressing both sense and antisenseRNA oligomers, an unexpected aberrantly sized product accumulates inlarge amounts (FIGS. 5A-D). Experiments with RNAse pretreatment of theRNAs prior to electrophoresis and blotting suggest that this largertranscript is double stranded. The ds RNA may be in the form of a simpleduplex, or could be covalently joined. Covalently linked siRNAs havebeen shown to be effective when expressed in cells, a result somewhatcontradictory to the results when using ex vivo delivered siRNAs(Elbashir, S. M. et al., 2001; Caplen, N. J. et al., 2001).

In order to ascertain whether or not there are differences in targetsite accessibilities for siRNA pairing as observed for antisense oligosand ribozymes (Scherr, M. et al., 1998; Scherr, M. et al., 2001;co-pending U.S. application Ser. No. 09/536,393, filed Mar. 28, 2000),two target sites for the siRNAs were tested. One site was chosen by anoligonucleotide library scanning mechanism designed to identify sitesaccessible to antisense pairing on native RNAs in cell extracts (Scherr,M. et al., 2001), whereas the other site was chosen at random in asegment of rev that overlaps with the HIV-1 tat sequence. Markeddifferences in accessibility to oligo pairing to these two sitestranslated to marked differences in siRNA inhibitory activities in therev-EGFP fusion. Despite the differences in potency against the rev-EGFPtarget, both siRNAs were potent inhibitors in HIV-1 co-transfectionassays. The invention thus demonstrates functional intracellularexpression of siRNAs in mammalian cells, particularly human cells.

An interesting result was the relationship between antisense DNAoligomer site-accessibility and the efficacy of the siRNAs targetedagainst the rev-EGFP transcript. More specifically, there was observed arelative lack of inhibition of the rev-EGFP target mediated by site IsiRNAs. This result was not the result of poor expression of theseoligomers, since they appear to be expressed in equivalent amounts tosite II siRNAs (FIGS. 5A-D). These differing results could be due to theposition of the siRNA target site relative to the end of the targettranscript, which has been demonstrated to limit amplification of thesiRNAs in Drosophila (Elbashir, S. M. et al., 2001). However, this doesnot seem to be the case since site I is positioned 301 nts downstream ofthe pIND-rev-EGFP transcriptional start site, which is well beyond theminimal distance required to amplify the siRNAs. These results could bedue to different targets used in different experiments. The relativeaccessibility of the site I in the context of the rev-EGFP fusion mRNAmay be limiting as shown by oligo-RNAseH experiments (FIG. 2). In theHIV-1 transcripts, site I is present in both the tat and revtranscripts, as well as in the singly spliced and unspliced transcripts.Given the complexity of different transcripts harboring site I, it isnot possible to state which of these is sensitive to the site I siRNAs.

Target mRNA for Testing siRNA.

A prerequisite for development of siRNA approaches to silence viral geneexpression is to have an appropriate human cell assay system. In orderto assay the siRNAs, rev was fused to EGFP (enhanced green fluorescentprotein) to provide a reporter system for monitoring siRNA function(FIG. 1A). Temporal control of target mRNA expression was obtained byinserting the rev-EGFP fusion gene in the Ecdysone-inducible pIND vectorsystem (Invitrogen) (FIG. 1A). It will be readily apparent to personsskilled in the art that alternative vector systems and promoters may beused for expressing selected target genes or target fusion genes duringco-transfection assays.

In order to use the inducible system, a 293/EcR cell line was used,which was engineered to respond to the insect hormone analoguePonasterone A. When the pIND-rev-EGFP vector was transfected into thesecells followed by addition of the inducer, EGFP fluorescence wasobserved as early as 3 hr after addition of ponasterone A and continuedfor more than 100 hr. In the absence of ponasterone A, EGFP fluorescencewas not observed.

In FIG. 1A, the relative locations of the two siRNA target sites in therev-EGFP target are indicated, as are the locations of these two targetsites in HIV transcripts from pNL4-3.

Target Site Accessibility in the Rev-EGFP Fusion Transcript.

It was previously demonstrated that synthetic DNA oligonucleotides incell or ovary extracts can be used to identify sites accessible to basepairing by both antisense DNA and ribozymes (Scherr, M. et al., 1998;Scherr, M. et al., 2001; Lee, N. S. et al., 2001). Semi-random 19-merDNA oligomer libraries (Scherr, M. et al., 2001) were used in cellextracts prepared from cells expressing the rev-EGFP mRNA transcripts.Using this approach to screen the entire rev sequence, only a singleTdPCR product was identified (data not shown), which centered within thesequence 5′GCCTGTGCCTCTTCAGCTACC 3′ (SEQ ID NO:10), located 213 ntsdownstream from the AUG codon of rev and 494 nts downstream of the siteof pIND transcription initiation. Since this sequence harbors a CUChammerhead ribozyme cleavage motif, a hammerhead ribozyme was alsosynthesized that cleaves after the CUC site. This enabled a comparisonbetween the inhibitory activity of the siRNA with a ribozyme expressedfrom the same promoter system. To determine whether or not there aredifferences in the siRNA mediated targeting of a given message, a second21 nt site with a 5′ G and 3′ C was selected, which has a total GCcontent similar to site I. The requirements for a 5′ G and 3′ C arebased on the first nucleotide of the pTZU6+1 transcript, which initiateswith a G (FIGS. 1C & D). The second target sequence,5′GCGGAGACAGCGACGAAGAGC3′ (SEQ ID NO:11), is also located in an exoncommon to tat and rev, 20 nts downstream of the rev translationalinitiation codon, and 301 nts downstream of the pIND transcriptionalinitiation signal.

In FIG. 1B, the schematic presentation of the upstream promoter andtranscript portion of the U6 expression cassette is shown with thesequences and depicted structure of the expected primary transcript. InFIG. 1C, the sequences of the 21 base sense and antisense inserts with astring of 6T's and XbaI are shown. The first G came from the mungbean-treated SalI of pTZU6+1 vector. The 6T's may be processed to the2T's (capital letters) by the Pol III RNA polymerase. In FIG. 1D, theputative siRNAs derived from co-expression of the sense and antisense 21mers (S/AS(I) or (I)), with 3′ UU overhangs are depicted.

To determine whether the two target sequences were equally accessible toantisense pairing, two 21-mer DNA oligonucleotides complementary to eachof the two siRNA target sites were synthesized and used as probes foraccessibility to base pairing with the rev-EGFP fusion transcripts incell extracts. It was demonstrated (FIG. 2) that site II is highlyaccessible to base pairing with its cognate oligo (89% reduction inRT-PCR product relative to the no oligo control). This was in contrastto the results obtained with the site I oligo, which reduced therev-EGFP transcript by 27% relative to the control. Since these twosites have marked differences in their accessibilities to antisensepairing (FIG. 2), they provided a good test for the role that targetaccessibility plays in siRNA-mediated targeting.

In FIG. 2, the ethidium bromide-stained bands represent RT-PCR productsfrom rev-EGFP (top, 673 nt) or beta Actin (bottom, 348 nt) mRNAs. Thelanes from left to right are: control, no added oligo, minus (−) or plus(+) RT; oligonucleotide probing for site I (−) or (+) RT;oligonucleotide probing of site II (−) or (+) RT. The reduction intarget mRNA is elicited by endogenous RNAse H activity as describedpreviously (Scherr, M. et al., 1998).

Genes encoding siRNAs targeted to site I or II were inserted behind thePol III U6 SnRNA promoter of pTZU6+1 (FIGS. 1B & C). The transcriptionalcassettes were constructed such that they are either in the same ordifferent vectors. The constructs in separate vectors provided a set ofsense and antisense controls.

Reduction of Target Gene Expression.

The siRNA sequences, along with sense, antisense, or ribozyme controlswere cotransfected with the target rev-EGFP expressing plasmid into293/EcR cells. Sixteen to twenty hours later, the inducer Ponasterone Awas added to the cell cultures resulting in induction of the rev-EGFPfusion product. The cells were incubated an additional 48 hours prior tofluorescent microscopic analyses and fluorescence activated cell sorting(FACS). Combined sense and antisense RNA oligomers targeted to site IIin the rev sequence reduced the EGFP signal by ca 90% relative to thecontrols, whereas the combined sense and antisense RNA oligomerstargeted against site I gave only a modest reduction in fluorescence(FIGS. 3 & 4). The inhibition mediated by the site II siRNAs was similarregardless of whether both sense and antisense RNA oligomers wereexpressed from the same plasmid or different plasmid backbones (seeFIGS. 1B & C). The control constructs, which included sense alone,antisense alone or a ribozyme targeted to site II, each expressed fromthe U6 promoter, gave no significant reduction of EGFP expressionrelative to the vector backbone control (FIGS. 3 & 4).

In FIG. 3, 293/EcR cells were co-transfected with pIND-rev-EGFP andvarious siRNA constructs as indicated. Cells were examinedmicroscopically for EGFP expression following Ponasterone A addition asdescribed herein. Panel E shows fluorescent cells after transfectionwith control which is an irrelevant sense/antisense construct to therev. Other types of controls (S(II), AS(II), vector) were similar (A, B,D). Panel C shows ˜90% reduction in fluorescent cells when 293/EcR cellswere transfected with S/AS(II). Panels F-J are DAPI-stained imagesshowing that the same number of cells are present in each field.Specific silencing of target genes was confirmed in at least threeindependent experiments.

In FIG. 4, 293/EcR cells were co-transfected with pIND-rev-EGFP andsiRNA constructs as described herein. Cells were analyzed for EGFPexpression by FACS and the level of fluorescence relative to cellstransfected with pIND-rev-EGFP alone was quantitated. Data are theaverage±SD of 3 separate experiments. Only the siRNA constructcontaining both sense and antisense sequences directed at accessiblesite II (S/AS(II) or S+AS(II)) showed approximately 90% reductionrelative to the controls or vector only. The various combinations of U6driven siRNA constructs co-transfected with pIND-rev-EGFP are indicated.S/AS indicates the vector with both sense and antisense siRNA sequencewhile S+AS indicates the siRNA sequences in separate vectors. Rbzindicates the hammerhead ribozyme against site II. Specific silencing oftarget genes was confirmed in at least three independent experiments.

Expression of siRNAs and Targets in 293 Cells.

Northern gel analyses were carried out to examine the expressionpatterns and sizes of the siRNAs transcribed from the U6 RNA Pol IIIpromoter system in 293 cells. These Northern gel analyses demonstratedstrong expression of sense and antisense RNAs as monitored byhybridization to the appropriate probes (FIG. 5).

In FIG. 5, RNA samples were prepared from 293/EcR cells transientlyco-transfected with pIND-rev-GFP and various siRNA constructs asindicated and subjected to Ponasterone A induction as described above.The total RNA was resolved on a 10% acrylamide/8M urea gel for siRNAs, a1% agarose/formamide gel for the target, or a 10% acrylamide/7M urea gelfor RNAse A/T1 treatment. In FIGS. 5A to 5D, hybridization was performedusing ³²P labeled DNA probes for sense or antisense transcripts for siteI and II siRNAs. The hybridizing products were ˜23- and ˜46-nts inlength. FIG. 5E shows the results of hybridization of the site IIdirected ribozyme (II) transcripts. RNAs prepared from cells expressingthe ribozyme for site II detected a transcript of the size expected forthe ribozyme transcript (˜75 nt). Since the probe used to detect theribozyme is also complementary to the antisense siRNA for site II, italso hybridized to the antisense RNAs targeted to site II.

The control RNAs (sense alone, antisense alone or ribozyme) were alldetected at the expected sizes (FIGS. 5A-E). RNAs prepared from cellssimultaneously expressing sense and antisense constructs generatedhybridized products of the sizes expected for the individual short RNAs(˜23 nts). In addition to the monomer sized RNAs, a strong hybridizationproduct approximately twice the size of the short RNA oligomers (˜46nts) was clearly visible (FIGS. 5A-E). This product was only detected inRNAs prepared from cells expressing both sense and antisense, andhybridized with both sense and antisense probes (FIGS. 5A-E). Since thegel system used to resolve the transcripts was a denaturing gel, itseemed unlikely that the aberrantly sized product could be dsRNA.Nevertheless, to test this possibility, the RNA samples were treatedwith a mixture of RNAse A and T1 prior to denaturing gel electrophoresisand blotting. Both of these RNAses preferentially cleave single strandedRNAs. Thus, if the aberrant product is double stranded, it should befully resistant to nuclease destruction. Two types of analyses wereperformed. The first involved simply treating the RNA samples with theRNAse mixture, whereas the second involved heating the samples to 95° C.prior to RNAse treatment (FIG. 5G).

In FIG. 5G, the RNAs from a combined transfection using S/AS(I+II) weretreated with a mixture of RNAse A and T1. Samples were either heated (+)or not heated (−) at 90° C. prior to RNAse treatment. The hybridizingproduct in the lane treated with RNAses, but without heat may be ˜21 ntsin length. This product was observed with probes for either sense orantisense siRNAs targeted to site II.

Fully duplexed RNAs should be resistant to cleavage, whereas heattreatment followed by quick cooling of the RNAs would separate thestrands and make them susceptible to RNAse cleavage. The resultsobtained (FIG. 5G) were consistent with the aberrantly sized productbeing double stranded. The non-heated sample treated with the RNAse mixgenerated a product migrating faster than the other RNAs. The fastermigrating RNA species hybridized to both the antisense and sense probes.In contrast, the sample that was heated prior to RNAse treatment gave nodetectable hybridizing bands. The faster migration of this product couldbe due to RNAse trimming of non-base paired ends of the duplex. Theseproducts could also derive from RNAse cleavage of single stranded loops,which would unlink the two RNAs, allowing for faster gel mobility.Importantly, the large, aberrantly sized transcripts are only generatedin cells expressing both sense and antisense transcripts, and thereforemust be dependent upon formation of ds RNAs (Bernstein, E. et al., 2001;Clemens, J. C. et al., 2000). In addition to the aberrantly sizedproduct, another band migrating between the aberrantly sized product andthe 106 nt U6 snRNA was detected only with a probe that is complementaryto the antisense siRNA for site II. This product was ˜65 nts in lengthand could be a product of the first dicer cleavage reaction on an RNAdependent RNA polymerase (RdRP) extended product of the siRNA antisense(Lipardi, C. et al., 2001; Sijen, T. et al., 2001). Finally, U6+1expression of the ribozyme targeted to site II (FIG. 5E) did not resultin inhibition of rev-EGFP expression (FIG. 4).

To determine whether siRNA complexes directed degradation of therev-EGFP mRNA, Northern hybridization analyses were carried out to probefor the rev-EGFP transcripts (FIG. 5F). FIG. 5F shows the results ofhybridization of the rev-EGFP fusion transcripts. Human GAPDH mRNA andU6snRNA were probed as internal controls for each experiment. These datademonstrated selective destruction of the fusion transcript only incells expressing the combination of site II sense and antisense siRNAs.The site I siRNAs, although abundantly expressed (FIGS. 5A and 5B)resulted in marginal inhibition of rev-EGFP expression (FIG. 4), withlittle degradation of the transcript (FIG. 5F). The combination of sitesI and II siRNAs resulted in less inhibition than the site II siRNAsalone. This is believed to result from a dosage effect in that theconcentration of each of the plasmids encoding these siRNAs was one halfthat used for the single site cotransfections. Most interestingly, thesite I siRNAs were potent inhibitors of HIV replication.

Inhibition of HIV by Expressed siRNAs.

In another embodiment of the invention, an siRNA expression system wasconstructed for inhibiting HIV-1 infection. In order to test thepotential inhibitory activity of the various constructs described above,each of the siRNA vectors (site I and II) as well as the controlconstructs were co-transfected with HIV-1 pNL4-3 proviral DNA into 293cells. At the intervals indicated in FIG. 6, supernatant samples werewithdrawn from the cell cultures and HIV-1 p24 viral antigen levels weremeasured. In FIG. 6, pNL4-3 proviral DNA was cotransfected with thevarious U6+1 driven siRNA constructs at 1:5 ratio of proviral DNA to U6construct DNAs. Twenty-four hours post transfection, and at theindicated times, supernatant aliquots were withdrawn for HIV-1 p24antigen assays. The various siRNA constructs used are indicated in FIG.6.

The site II siRNAs were found to strongly inhibit HIV-1 replication inthis assay. Somewhat unexpectedly, the site I siRNAs also potentlyinhibited HIV-1 replication (as measured by p24 antigen production). Thecombination of both site I and II siRNA constructs was the most potent,providing ca four logs of inhibition relative to the control constructs.Such potent inhibition of HIV-1 has not been previously observed withother RNA-based anti-HIV-1 agents using a co-transfection assay.Possible explanations for the differences in inhibitory activity by thesite I siRNAs against the rev-EGFP fusion versus HIV-1 itself areaddressed herein. The observation that two different HIV targets areboth substrates for siRNA is highly encouraging for strategies requiringmultiple targeting to circumvent genetic resistance of the virus.

The present invention is further illustrated by the following exampleswhich are not intended to be limiting.

Example 1 Accessibility Assay

To evaluate the accessibility of target sequences for antisense basepairing, endogenous RNase H activity present in the cell extractsprepared from the stable 293 cells containing the rev-GFP was utilized.Two DNA oligonucleotides complementary to each of the two target siteswere synthesized and used as probes for accessibility in cell extractsaccording to the protocol of Scherr, M. et al., 1998, as describedherein, with some minor modifications.

Stable 293 cells containing CMV-revGFP gene were grown to 50 to 90%confluency in 100 mm dishes. Cells were harvested using a cell scraperand rinsed two times in phosphate buffered saline (PBS) twice. The cellswere resuspended in the same volume of hypotonic swelling buffer (7 mMTris-HCl, pH 7.5, 7 mM KCl, 1 mM MgCl₂, 1 mM β-mercaptoethanol) and1/10th of the final volume of neutralizing buffer (21 mM Tris-HCl, pH7.5, 116 mM KCl, 3.6 mM MgCl₂, 6 mM β-mercaptoethanol) on ice. Theresuspended solutions were sonicated 15 seconds three times in an icewater bath. The homogenate was centrifuged at 20,000×g for 10 minutes at4° C. The supernatants were used immediately, or stored as aliquots inthe same volume of glycerol storage buffer (15 mM Tris-HCl, pH7.5, 60 mMKCl, 2.5 mM MgCl₂, 45% glycerol, 5 mM β-mercaptoethanol) at −80° C.These frozen aliquots were always used within 3 months post freezing.

The cell extracts were incubated with 4 μM of the respective 21 bpantisense oligodeoxyribonucleotides (site I or II) for 15 minutes at 37°C. in a total volume of 30 μl cleavage buffer (100 mM Tris-HCl, pH7.5,100 mM MgCl₂, and 10 mM DDT). After phenol extraction and ethanolprecipitation, the precipitates were digested with 20 U of DNase I for45 minutes at 37° C., followed by phenol extraction and ethanolprecipitation. The precipitates were resuspended in DEPC water andmonitored for OD₂₆₀ absorption.

The reverse transcriptase (RT) reaction was carried out using 300 ng to1 μg total RNA prepared from the above 30 μl extract sample with 5 U ofMoloney murine leukemia virus reverse transcriptase (Mo-MLV Rtase, LifeTechnologies, Inc. NY) according to the manufacturer's instructions.

First-strand priming was performed with 20 pmol of 3′ primer of an oligocomplementary to the rev sequence or 50 ng of random hexamer primers.β-actin was used as an internal control. PCR reactions for each set ofprimers were performed separately in a total volume of 50 μl containing10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl₂, 0.1 mM of each dNTP,0.4 μM of each primer, 1.5 U of taq DNA polymerase and 2 μl of RTreaction sample. The PCR was carried out at 94° C. for 30 seconds, 68°C. for 30 seconds, and at 72° C. for 30 seconds for a total of 24 to 36cycles after cycle studying of control.

Reaction samples were separated on a 1.2% agarose gel and visualized byethidium bromide staining, then quantified using AlphaImager™quantitation software (Alpha Innotech Corp.).

Example 2 Constructs

A SacII (filled in)-EcoRI fragment containing the rev-GFP fusion gene ofCMV-rev-GFP was inserted into HindIII (filled in)-EcoRI sites of thepIND vector (Invitrogen), yielding the pIND-rev-GFP construct of (FIG.1A).

To construct the siRNA expression vectors, two cassettes were preparedusing the pTZ U6+1 vector (FIG. 1B). (Bertrand, E. et al., 1997; Good,P. D. et al., 1997). One cassette harbors the 21-nt sense sequences andthe other a 21 nt antisense sequence. These sequences were designed totarget either site I or site II (FIG. 1A). A string of 6T's was insertedat the 3′ terminus of each of the 21 mers followed by a XbaI restrictionsite. The 20-nt sense or antisense sequences (excluding the first G)with a string of 6T's and the XbaI restriction site were prepared fromsynthetic oligonucleotides (Lee, N. S. et al., 2001; Lee, N. S. et al.,1999). The first G of the inserts was provide by the Sal I site of thevector which was rendered blunt-ended by mung bean nuclease. The insertswere digested by BsrBI for sense and AluI for antisense (site I), StuIfor sense and SnaBI for antisense (site II) for blunt end cloningimmediately downstream of the U6 promoter sequence. The 3′ ends of theinserts were digested with Xba I for insertion in the Sal I(blunted)-Xba I digested pTZU6+1 vector to create the desiredtranscription units (FIG. 1C).

To create plasmids in which both sense and antisense sequences were inthe same vector, the pTZU6+1 sense sequence harboring vectors weredigested with BamHI (which was filled in using T4 DNA polymerase) andHindIII. The digested fragments containing the sense sequences weresubcloned into the SphI (filled in by T4 DNA polymerase)-HindIII sitesof the antisense AS(I) or AS(II) constructs, generating both sense andantisense transcription units (S/AS(I) or S/AS(II)) (FIG. 1C). The DNAsequences for each of the above constructs were confirmed prior to use.

Example 3 Cell Culture

293/EcR cells were grown at 37° C. in EMEM supplemented with 10% FBS, 2mM L-glutamine and 0.4 mg/ml of Zeocine. Twenty-four hour beforetransfection, cells were replated to 24- or 6-well plates at 50-70%confluency with fresh medium without Zeocine. Co-transfection of targetplasmids (pIND-rev-GFP) and siDNAs was carried out at 1:1 ratio withLipofectamine Plus™ reagent (Life Technologies, GibcoBRL) as describedby the manufacturer. 0.5 mg pIND-rev-GFP and 0.5 mg siDNAs and 0.1 mgpCMV-lacZ (for transfection efficiency), formulated into LipofectaminePlus, were applied per 6-well culture. Cells were incubated overnightand on the following day 5 mM Ponasterone A (Invitrogen) was added toinduce expression of pIND-rev-EGFP. Two days post induction thetransfected cells were harvested to measure EGFP fluorescence by FACSusing a modular flow cytometer (Cytomation, Fl). Transfectionefficiencies were normalized using a fluorescent b-galactosidase assay(Diagnostic Chemicals Ltd, CN). Fluorescent microscope imaging was alsoperformed to monitor EGFP expression. For the microscopic visualization,cells were grown on glass coverslips in 24-well plates. Co-transfectionswere carried out on glass coverslips using 0.25 mg each of rev-EGFP andsiRNA expression plasmid DNAs (total 0.5 mg). After 2 days, transfectedcells were fixed in 4% PFA for 15 minutes at room temperature andtreated with antifading reagent containing DAPI. Images were collectedusing an Olympus BX50 microscope and a DEI-750 video camera (Optronics)at 40× magnification with exposure time of ⅛ sec. Specific silencing oftarget genes was confirmed in at least three independent experiments.

Example 4 Northern Blotting

RNA samples were prepared from 293-EcR cells transiently co-transfectedwith pIND-rev-GFP and siRNAs and subjected to Ponasterone A induction asdescribed above. Total RNA isolation was preformed using the RNA STAT-60(TEL-TEST “B”) according to the manufacturer's instruction. The totalRNA was resolved on a 10% acrylamide/8M urea gel for siRNAs or a 1%agarose/formamide gel for the target, and transferred onto Hybond-N⁺membrane (Amersham Pharmacia Biotech). The hybridization and wash stepswere performed at 37° C. To detect the sense or antisense siRNAs,radiolabeled 21-mer DNA probes were used. Human U6 snRNA and GAPDH mRNAwere also probed for as internal standards. To detect the ribozymetargeted against site II of the rev mRNA, a 42-mer probe complementaryto the entire ribozyme core and flanking sequences was used (this probealso detects the antisense siRNA oligomer for site II). Forcharacterization of the aberrantly sized 46-nt RNAs, total RNAs fromS/AS(I+II) were treated with a mixture of RNAase A and T1 for 30 minutesat 37° C. either with or without preheating at 90° C. for 5 minutes.

For detection of the rev-EGFP mRNA, a 25-mer probe complementary to theEFP mRNA of the rev-GFP fusion protein was used.

Example 5 HIV-1 Antiviral Assay

For determination of anti-HIV-1 activity of the siRNAs, transient assayswere performed by cotransfection of siDNAs and infectious HIV-1 proviralDNA, pNL4-3 into 293 cells. Prior to transfection, the cells were grownfor 24 hours in six-well plates in 2 ml EMEM supplemented with 10% FBSand 2 mM L-glutamine, and transfected using Lipofectamine Plus™ reagent(Life Technologies, GibcoBRL) as described by the manufacturer. The DNAmixtures consisting of 0.8 μg siDNAs or controls, and 0.2 μg pNL4-3 wereformulated into cationic lipids and applied to the cells. After 1, 2, 3and 4 days, supernatants were collected and analyzed for HIV-1 p24antigen (Beckman Coulter Corp). The p24 values were calculated with theaid of the Dynatech MR5000 ELISA plate reader (Dynatech Lab Inc). Cellviability was also performed using a Trypan Blue dye exclusion count at4 days after transfection.

The above demonstrates the invention's utility for, among other things,designing and testing siRNA transcripts for a variety of genetic andtherapeutic applications. The invention also is believed to demonstratefor the first time the functional expression of siRNAs in mammaliancells.

The above results also demonstrate the utility of siRNAs as HIV-1inhibitory agents. By combining several siRNA constructs targeted todifferent sites in the HIV-1 genome, it should be possible to circumventgenetic resistance of the virus, thereby creating a potent gene therapyapproach for the treatment of HIV-1 infection.

The publications and other materials cited herein to illuminate thebackground of the invention and to provide additional details respectingthe practice of the invention are incorporated herein by reference tothe same extent as if they were individually indicated to beincorporated by reference.

While the invention has been disclosed by reference to the details ofpreferred embodiments of the invention, it is to be understood that thedisclosure is intended in an illustrative rather than a limiting sense,as it is contemplated that modifications will readily occur to thoseskilled in the art, within the spirit of the invention and the scope ofthe appended claims.

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What is claimed is:
 1. A method for testing the expression and functionof a small interfering RNA (siRNA) molecule, comprising: co-introducinginto a mammalian cell (a) one or more vectors comprising (i) a firstcassette comprising an RNA pol III promoter operatively linked to afirst nucleic acid encoding a sense strand of a double stranded siRNAmolecule and (ii) a second cassette comprising an RNA pol III promoteroperatively linked to a second nucleic acid encoding an antisense strandof the double stranded siRNA molecule, wherein the first nucleic acidcomprises a DNA sequence encoding the sense strand positioned at thefirst nucleotide of the transcript of the RNA pol III promoter and thesecond nucleic acid comprises a DNA sequence encoding the antisensestrand positioned at the first nucleotide of the transcript of the RNApol III promoter, wherein the first cassette and the second cassette arein a same vector or are in separate vectors and wherein the doublestranded siRNA molecule is specific for a target sequence in a targetgene, and (b) a vector comprising the target gene and a suitable secondpromoter; growing the mammalian cell under conditions suitable forexpression of the nucleic acid sequences and the target gene;determining if the double stranded siRNA molecule is expressed and ifexpression of the target gene is reduced, whereby the expression andfunction of the siRNA molecule is tested.
 2. The method of claim 1,wherein the mammalian cell is a human cell.
 3. The method of claim 1,wherein the nucleic acid sequences are in separate vectors.
 4. Themethod of claim 1, wherein the nucleic acid sequences are in the samevector.
 5. The method of claim 1, wherein at least one of the vectors isa plasmid vector.
 6. The method of claim 1, wherein at least one of thevectors is a viral vector.
 7. The method of claim 1, wherein the RNA polIII promoter is a mammalian U6 poly III promoter.